PROJECT SUMMARY/ABSTRACT The placenta is central to embryonic/fetal development of all eutherian species including humans. However, much remains unknown about the diversity, the ontogeny, and the molecular nature of the cells that make up the placenta. The placenta also represents one of the most rapidly evolving organs as exemplified by the its highly distinct structure in mouse and human. The changes in structure arose from a combination of divergent and convergent evolution that cannot be understood by histology alone. Understanding the analogous cell types and molecular programs between species will greatly improve the ability to translate findings from the mouse model system to humans. A long-term goal of the lab is to understand the pathways that regulate placental development from placentation to delivery and determine how these pathways become misregulated in placental diseases such as pre-eclampsia, intrauterine growth retardation, and pre-term birth. Our immediate goal is to better understand the diversity of cell types and cross-species relationships in function between those cell types in mouse and human. Our particular focus in this grant is on the cells that give rise to and constitute the exchange membrane existing between maternal and fetal blood. Across this surface, there is the essential exchange of nutrients, gases, wastes, and signaling molecules. Defects in this membrane are highly detrimental to the developing embryo. During the previous cycle of this center grant, our laboratory discovered that an eutherian-specific microRNA cluster is essential for the normal development of the exchange membrane within the labyrinth of the mouse placenta. Here, we will expand on these findings by first molecularly defining the cell types that exist within the labyrinth, determining how those cell types are impacted at the molecular level by the loss of the miRNA cluster, and how those findings can be translated to the analogous structure in the human placenta. We address these questions in three aims. In aim 1, we will use single nucleus RNA sequencing to determine the cell types and lineage relationships of the cells within the mouse labyrinth including the individual nuclei of the syncytiotrophoblast layer. In aim 2, we will use combination of sorting, single cell sequencing, and in vitro experiments to uncover the impact of miR-290 on the cellular composition and transcriptional heterogeneity of labyrinth trophoblasts. In aim 3, we will use single cell nucleus RNA sequencing, cutting edge bioinformatics approaches, and in vitro manipulation of isolated cells of the human placenta to translate the findings in mouse to human. In addition to dissecting the role of a eutherian-specific microRNA cluster in placental development, successful completion of the proposed experiments will impact our understanding of cellular composition and ontogeny of the key exchange structures of the mouse and human placenta and provide a paradigm on how to translate mouse genetic/molecular findings to human placentation and placental diseases.